Genetic Manipulation of Paradiplonema papillatum

Here we present a collection of methods established for genetic manipulation of Paradiplonema papillatum (also referred to as Diplonema papillatum), so far the only diplonemid species that is genetically tractable and can therefore serve as a model organism, enabling functional studies of this very abundant and species-rich group of marine protists. We describe a complete transformation protocol which includes: (1) the Alamar blue assay, a method that serves to identify antibiotics, to which P. papillatum is sensitive and can be used for transformant selection; (2) the design and preparation of DNA constructs for functional genomics; and (3) two protocols for electroporation and transformant screening. We conclude with an immunoprecipitation protocol optimized for pulling down target-tagged proteins along with their interaction partners, allowing the characterization of protein complexes.